The overall objective is to construct and optimize novel human CEA-based DNA vaccines for the effective immunotherapy of colon carcinoma. The investigators will test the hypothesis that peripheral T cell tolerance to these tumor self-antigens can be overcome by DNA vaccines boosted by effective adjuvants designed to generate cytolytic T lymphocyte (CTLs) specific for CEA epitopes expressed as MHC class I complexes on colon carcinoma cells. Emphasis will be on optimizing antigen processing and presentation in mouse models either transgenic for CEA or double transgenic for CEA and HLA-A2.1Kb. Their aim is to use such models for optimization of vaccine by antibody-cytokine fusion proteins and to investigate basic concepts such as mechanisms of T cell co-stimulation, generation of tumor-specific CTLs and T memory cells and establish principles for adoptive immunotherapy. The specific aims designed to achieve these objectives are: 1) construction of optimal human CEA-specific DNA vaccines containing first the entire CEA gene and then minigenes encoding specific CEA peptides with HLA-A*0201 anchor residues. Delivery of the vaccines by injction of naked KNA or orally by galvage using attenuated strains of either Salmonella typhimurium or Listeria monocytogenes; 2) optimization of antigen processing in the 20S proteasome and presentation by using ubiquitinated versions of the entire CEA gene, minigenes encoding several CEA nonapeptides organized as as a string of beads or direct targeting of single CEA or repeat epitopes to the endoplasmic reticulum; 3) achievement of optimal adjuvanticity using either unmethylated CpG dinucleotide motifs or CD40 Ligand/Trimer co-expression; and 4) determination whether antibody-IL2 fusion proteins can effectively boost DNA vaccines to achieve optimal, long-lived tumor-protective immunity, as well as eradicate established metastases, and identification of immunological mechanisms involved in generating tumor-specific CTLs and T memory cells. The achievement of this proposal's objectives should lead to the design of effective DNA vaccines based on rational immunological principles that may ultimately lead to the improved treatment of colon cancer.